5-hydroxytryptamine in the carotid body of the cat.

Glomus cells, at least in the carotid body of cats, contain catechol and indolamines. Cells containing adrenaline, noradrenaline, and 5-hydroxytryptamine were identified.

Relationship of prolactin release in lactating rats to milk ejection, sleep state, and ultrasonic vocalization by the pups.

PRL release, milk ejection, and electroencephalographic states of sleep were monitored in conscious, lactating Holtzman rats. Plasma PRL levels were low in rats separated from their litters, but they increased when pups were returned to the cage, attached to the nipples of the mother, and suckled. The pups emitted ultrasonic vocalizations upon their return to the cage and before their attachment to the nipples. Exposure of mothers to the vocalizations of pups, whereas nipple attachment was prevented, failed to increase PRL release. In confirmation of previous work, milk ejection did not occur until the suckled mother exhibited electroencephalographic signs of sleep, and milk ejection could be inhibited if sleep was prevented. In contrast, sleep was not a prerequisite for PRL release and sleep deprivation of suckled mothers could not inhibit release of PRL. In summary, suckling-induced release of PRL was followed by an increased incidence of sleep and then by milk ejection. It is hypothesized that the rise in plasma PRL induces the sleep necessary for the reflex release of oxytocin required for milk ejection.

Milk ejection reflex linked to slow wave sleep in nursing rats.

Correlations between cerebral activity of nursing rats and the milk ejection reflex were studied in Sprague-Dawley rats with 15- to 17-day-old litters. The stretch reaction of the pups, which expresses the onset of milk ejection, was closely correlated with the slow sleep epochs of the mother. Once the litter started suckling, milk ejection only took place when the mother fell asleep and electroencephalographic features of slow wave sleep appeared. Milk ejection was never found during paradoxical sleep nor when the mother was awake. Sleep deprivation for 30 min impaired milk ejection in spite of continuous suckling of the nipples by the pups. If the mother was allowed to sleep immediately afterwards, ejection of milk occurred. A 24-h sleep-wakefulness pattern did not show differences between nursing and controls. Our results show that suckling, although necessary, is not enough to set off milk ejection. This reflex only appears when the mother falls asleep, suggesting that oxytocin release is linked to suckling and slow wave sleep.

Changes in adenohypophysial dopamine related to prolactin release.

In order to study whether changes in adenohypophysial dopamine are only related to PRL release or also to the variation of LH or FSH, dopamine (DA) in the pars distalis was measured during the estrous cycle and after castration of both male and female rats. Catecholamines were assayed using a highly sensitive radioenzymatic method. Dopamine and norepinephrine were also measured in the median eminence of the same rats. Adenohypophysial dopamine fluctuates during the estrous cycle, reaching its lowest levels on the afternoon of proestrus. Ovariectomy increases the DA content of the anterior lobe and estrogens reverse this effect. Orchidectomy does not change the amount of DA in the anterior pituitary. DA concentration in the median eminence parallels the changes observed in the pars distalis. We conclude that anterior pituitary dopamine concentration is closely related to PRL release and is not associated with changes in the release of LH or FSH.

Acute changes in norepinephrine content in the median eminence induced by orchidectomy or testosterone replacement.

Median eminence catecholamines were measured in individual samples from intact, castrated, or sham-operated male rats, in order to correlate changes in catecholamine content with the early post-castration rise in serum LH levels. Both norepinephrine (NE) and dopamine (DA) were measured by an enzymatic-isotopic assay and advantage was also taken of a method recently developed that makes it possible to obtain the median eminence free of the surrounding hypothalamus. Determinations were made at 4, 8, or 24 h after surgery. All animals were killed between 1400 and 1600 h. It was observed that castration increases the ME content of NE significantly at all the times studied, with peak values 8 h after orchidectomy. Sham operation induced a significant decrease in NE content in the ME 4 h after surgery. By 8 h the NE values had returned to intact control levels. Both castration and sham operation induced a significant decrease in DA levels 4 h after surgery. By 8 h, DA values had returned to intact control levels in both groups. When the NE levels in castrated rats had reached the highest value, serum LH values had not departed from control levels, indicating that the change in NE content in the ME takes place prior to the expected rise in LH. Testosterone replacement therapy (40 mug/100 gBW) of castrated rats blocked the increase in NE content observed 8 h after castration. Dopamine levels were not affected by this treatment. These results support the concept that the NE contained in median eminence catecholaminergic terminals participates in the feed-back regulation of LH secretion in the male rat, while DA is apparently not involved under the present conditions.

Involvement of adenohypophysial dopamine in the regulation of prolactin release during suckling.

Dopamine (DA) was measured in the anterior lobe of the pituitary and median eminence from lactating rats. The effect of pup separation and suckling was studied in order to correlate changes in DA levels with changes in serum PRL. In lactating rats separated from their pups, low levels of circulating PRL were found at 2,4, and 8 h. DA levels in the median eminence showed a decline at 2h; at 4 and 8 h of separation, a significant increase was observed. In the pars distalis, the concentration of DA increased with the length of the nonsuckling interval. Suckling induced a rapid rise in serum PRL levels in rats that were separated from their pups 4 h earlier. Under these conditions, a significant decrease in DA levels in the median eminence and pars distalis was observed as early as 5 min after the onset of suckling; at 30 min, the DA levels were still low. In the situations studied (suckling and pup separation), a negative correlation between serum PRL and DA levels in both the median eminence and pars distalis was always found.

Serotonergic terminals in the anterior hypothalamic nucleus involved in the prolactin release during suckling.

The present studies were designed to localize within the hypothalamus and neighboring areas the serotonergic terminals which are implicated in suckling-induced PRL release. The initial experiments were performed to characterize the circulating hormone profile induced by suckling in lactating rats, previously separated from their pups. Five minutes of suckling induced an increase in serum PRL only. During these 5 min, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid concentrations were determined in the pars nervosa of the pituitary gland, hypothalamic nuclei, dorsal, and median raphe nuclei. An increase by 80% (P less than 0.01) in 5-HT concentration was found only in the rostral part of the anterior hypothalamic nucleus (rNHA). In order to investigate causal effect between the altered 5-HT neuronal activity in the rNHA and the suckling-induced PRL release, serotonergic neurotoxin was bilaterally injected in the rNHA on day 1 of lactation. Litters were adjusted to eight pups each and weighed daily to determine litter growth rates. On day 8 of lactation, litters were separated from their mothers for 4 h and allowed to suckle for 5 or 15 min after which the mothers were decapitated. Litters from lesioned animals grew at a lower rate (P less than 0.0001) than control and sham-operated animals. Serum PRL increased with suckling in animals bearing the correct rNHA lesions, but the values were lower than in control and sham-operated animals after 5 (P less than 0.05) and 15 (P less than 0.01) min. Therefore we postulate that the rNHA is the site of termination of a stimulatory serotonergic pathway on PRL release induced by suckling.

Differential effects of superior and inferior spermatic nerves on testosterone secretion and spermatic blood flow in cats.

It has been postulated that testosterone secretion is partially regulated by signals from the spermatic nerves. To further examine this hypothesis in vivo, the superior (SSN) or the inferior (ISN) spermatic nerves were stimulated electrically (varying intensity, 25 Hz, 0.2 msec, 10 min) in anesthetized cats, determining the testosterone concentration and the blood flow in the spermatic vein. In some additional experiments arterial blood was sampled, and norepinephrine (NE) output was calculated. Stimulation of the SSN (25-35 V) increased the testosterone concentration in spermatic vein blood (P < 0.01 compared with prestimulation levels). The response varied among animals, reaching a 50-100% increase in some animals, whereas in others it ranged from almost undetectable to more than 10 ng/100 g x min. Under the same experimental conditions, the NE output increased from 135.4 +/- 99 to 1614.2 +/- 347 pg/ml (P < 0.01), and spermatic blood flow decreased from 24.1 +/- 1.42 to 20.2 +/- 1.65 ml/min x 100 g (P < 0.05) during nerve stimulation. By contrast, stimulation of the ISN (25-35 V) modified neither the testosterone concentration, the NE output, nor the blood flow in the spermatic vein. High intensity stimulation (36-70 V) of each spermatic nerve evoked different vascular and hormonal effects. SSN activation induced a marked decrease in spermatic blood flow during stimulation and an increase in the testosterone response, whereas ISN activation resulted only in an enhanced spermatic blood flow. Our results suggest that testosterone secretion, although mainly dependent on gonadotropin secretion, could be further regulated by neural inputs from the SSN acting directly or alternatively through changes in blood flow. It would appear that the SSN mainly supplies the vasoconstrictor fibers to the testis, whereas the ISN provides vasodilator fibers.

Electronmicroscopic and electrophysiological studies of the teat branch of the XIII thoracic nerve: relationship with lactation in the rat.

Electrical stimulation of the XIII thoracic nerve (the 'mammary nerve') causes milk ejection and the release of prolactin and other hormones. We have analysed the route of the suckling stimulus at the level of different subgroups of fibres of the teat branch of the XIII thoracic nerve (TBTN), which innervates the nipple and surrounding skin, and assessed the micromorphology of the TBTN in relation to lactation. There were 844 +/- 63 and 868 +/- 141 (S.E.M.) nerve fibres in the TBTN (85% non-myelinated) in virgin and lactating rats respectively. Non-myelinated fibres were enlarged in lactating rats; the modal value being 0.3-0.4 micron 2 for virgin and 0.4-0.5 micron 2 for lactating rats (P greater than 0.001; Kolmogorov-Smirnov test). The modal value for myelinated fibres was 3-6 micron 2 in both groups. The compound action potential of the TBTN in response to electrical stimulation showed two early volleys produced by the A alpha- and A delta-subgroups of myelinated fibres (conduction velocity rate of 60 and 14 m/s respectively), and a late third volley originated in non-myelinated fibres ('C') group; conduction velocity rate 1.4 m/s). Before milk ejection the suckling pups caused 'double bursts' of fibre activity in the A delta fibres of the TBTN. Each 'double burst' consisted of low amplitude action potentials and comprised two multiple discharges (33-37 ms each) separated by a silent period of around 35 ms. The 'double bursts' occurred at a frequency of 3-4/s, were triggered by the stimulation of the nipple and were related to fast cheek movements visible only by watching the pups closely. In contrast, the A alpha fibres of the TBTN showed brief bursts of high amplitude potentials before milk ejection. These were triggered by the stimulation of cutaneous receptors during gross slow sucking motions of the pup (jaw movements). Immediately before the triggering of milk ejection the mother was always asleep and a low nerve activity was recorded in the TBTN at this time. When reflex milk ejection occurred, the mother woke and a brisk increase in nerve activity was detected; this decreased when milk ejection was accomplished. In conscious rats the double-burst type of discharges in A delta fibres was not observed, possibly because this activity cannot be detected by the recording methods currently employed in conscious animals.(ABSTRACT TRUNCATED AT 400 WORDS)

An uneven distribution of choline acetyltransferase activity in the pituiatry neurointermediate lobe of the rat.

The neurointermediate lobe of the pituitary gland of male rats was assayed for choline acetyltransferase (CAT) activity. Precise microsample punches were obtained from neurohypophysial tissue, pure pars intermedia tissue and from the junction area between them. The level of CAT activity (pmol/h per microgram protein) in the neurohypophysis, pars intermedia and junction area were 0.390 +/- 0.038 (S.E.M.), 0.228 +/- 0.042 and 1.824 +/- 0.268 respectively. These values show an uneven distribution of CAT in the neurointermediate lobe. The hypothesis of a cholinergic system located in the junction area has been advanced.

Dopamine enters lactotrophs and reaches their secretory granules.

The presence of dopamine in the lactotroph cell, as well as in isolated prolactin secretory granules, was demonstrated by means of an histochemical reaction for electron microscopy. Biochemical assays further confirmed the presence of dopamine in the secretory granules. Autoradiographic preparations examined by light microscopy showed dopamine internalization in dispersed anterior pituitary cells. Isolated anterior pituitary lactotroph cells incorporated more [3H]dopamine than a fraction containing other anterior pituitary cells.

Serotonin induces gonadotrophin release through stimulation of LH-releasing hormone release from the median eminence.

The effects of serotonin (5-HT) on the release of gonadotrophins and LH-releasing hormone (LHRH) were examined in an in-vitro perifusion system using median eminences and/or anterior pituitaries obtained from male or pro-oestrous female rats. Animals were killed by decapitation between 12.00 and 13.00 h. A serial double-chamber perifusion system was employed. Three types of experiments were performed. In the first, median eminences were placed in the first chamber and one anterior pituitary in the second chamber. In the second group, only the anterior pituitary was perifused. In the third group, only five median eminences were perifused. In the first and second experiments, LH, FSH and prolactin were determined in the perifusion efflux by radio-immunoassay (RIA). In the third experiment, LHRH was determined by RIA. Addition of 5-HT (final concentrations 0.06, 0.6 and 6.0 mumol/l) into the first chamber containing the median eminences stimulated the release of LH and FSH from the pituitary, but did not affect the levels of prolactin in the effluent in the same experiment (pro-oestrous rats). The stimulatory effect of 5-HT was blocked by the addition of cyproheptadine (l mumol/l) in the perifusion fluid. The introduction of 5-HT (0.6 mumol/l) into the tube connecting the first and second chambers did not modify the release of LH, nor did 5-HT added to the pituitaries perifused alone.(ABSTRACT TRUNCATED AT 250 WORDS)

NOS is present in the brain of Triatoma infestans and is colocalized with CCK.

Immunohistochemistry was used to establish the presence of nitric oxide synthase (NOS) in the central nervous system of Triatoma infestans which is the main vector for Chagas' disease in Argentina and neighbouring countries. In addition, we have investigated the presence of cholecystokinin (CCK) and studied the possible coexistence of these molecules. The results show NOS-like immunoreactivity (LI) in neurones of the soma rind of the protocerebrum, the optic lobe and in the lateral part of the sensory deutocerebrum with a few cells in the suboesophageal and the prothoracic ganglia. The distribution of CCK-LI was similar to that of NOS-LI and in several areas both molecules coexisted in neurones and fibres. The results suggest that nitric oxide may act as a neurotransmitter in the brain of insects.

Dispersion and culture of magnocellular neurons from the supraoptic nucleus of the adult rat.

A simple and efficient procedure for the dispersion and culture of magnocellular neurons of the adult rat hypothalamus was developed. The enzymatically and mechanically dispersed cells were highly viable and showed neurite outgrowth after 3 days of culture. The neurons could be maintained for more than 4 weeks without any sign of deterioration. Light-optic immunocytochemistry of the cultured cells revealed the presence of many oxytocin- and vasopressin-immunoreactive neurons. The results indicate that the method may be suitable for many experimental purposes.

Sympathetic innervation of the median eminence.

Bilateral removal of the superior cervical ganglia (SCG) decreases the adrenaline (NE) content of the median eminence. Bilateral removal of the SCG and bilateral deafferentation of the medial basal hypothalamus (MBH) together induce a greater decrease of NE content of the median eminence than that resulting from either one of the operations performed alone. Our results indicate that the median eminence receives a noradrenergic innervation arising from the SCG and that the SCG is the main origin of the NE remaining in the median eminence after bilateral deafferentation of the MBH.

Origin and termination of dorsal raphe-median eminence projection.

The origin and termination of the dorsal raphe (DR)-median eminence (ME) projection were studied by combining well-limited small electrolytic lesions of different parts of the DR, the axonal degeneration and the serotonin content in the median eminence. Our results show that the rostral third of the DR is a source of serotonergic innervation to the ME and that the rest of the nucleus does not contribute to this projection. The light microscopy study revealed that degenerating fibers occupy the internal layer of the ME. These results were confirmed by electron microscopy which in addition showed degenerating terminals.

Central connections of the toad neural lobe as shown by retrograde neuronal labeling: classical and new connections.

Iontophoretic deposit of horseradish peroxidase (HRP) in the hypophysial neural lobe (NL) of the toad labeled neurons in the classical neurosecretory preoptic magnocellular nucleus. Further, a number of labeled neurons appeared in the whole extension of the periventricular and medial preoptic nuclei. The latter were continuous dorsally with two other labeled areas known as the bed nucleus of the hippocampal commissure (which appears to be homologous to the subfornical organ in mammals) and the ventromedial thalamic area of Frontera. The possible functional role of many of these afferents to the neural lobe is unknown because all types of neurons were labeled in the projecting zones and, in addition, many are outside of the generally recognized neurosecretory groups.

Acute superior cervical ganglionectomy depresses the postcastration rise of gonadotropins in male rats.

In male rats, superior cervical ganglionectomy (SCGx) delayed the rise of serum FSH and LH induced by orchidectomy by 24 h. SCGx resulted in a decrease of median eminence norepinephrine (NE) content 16 h after surgery and in an increase of medio-basal hypothalamic cAMP synthesis and receptor occupancy. These data indicate that NE release from degenerating terminals originating in the superior cervical ganglion neurons modifies the regulatory mechanisms controlling the rise of gonadotropins after orchidectomy in rats.

Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat.

Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

Afferent connections of the hypothalamic retrochiasmatic area in the rat.

The afferent projections to the retrochiasmatic area (RCA) of the rat hypothalamus have been studied by means of retrograde axonal transport of horseradish peroxidase. Iontophoretic deposit of the marker was used in most animals, and consistent projections from the hypothalamic ventromedial nucleus, the lateral part of the substantia nigra and the parabigeminal nucleus (NPB) were observed. A comparison between NPB projections and projections from the neighboring tegmentum suggests that some neurons of the NPB project to both the RCA and the superior colliculus. Consequently, these NPB neurons might link visual information on its way toward the retrochiasmatic area which, in view of its strategic position, could play a role in neuroendocrine processes.